DE602005004902T2 - Artificial protein, method for absolute quantification of proteins and its use - Google Patents

Description

Field of the invention

The The present invention relates to proteomics, and more particularly to absolute quantification of proteins.

Background of the invention

Of the The need for absolute quantification is increasing in proteomics urgent. The most promising method is based on more stable Isotope dilution, that the simultaneous determination of representative proteolytic Peptides and stable isotope labeled analogs includes. The basic limitation, which precludes a far-reaching implementation of this approach, is the availability of Standard signature peptides in exactly known amounts.

The both primary Topics in proteomics are the protein identification and the Comparison of protein expression levels in two physiological or pathological conditions (comparative proteomics). The long-term goal, able to to define the entire proteome of a cell is still not realized, but the characterization of many thousands of proteins in a single analysis is now possible.

In order to the proteomics becomes a platform technology that evolves to the developing one Field of systems biology, there is a crushing Need to improve quantification (Righetti, Eur J Mass Spectrom 10 (2004), 335-348). Most comparative proteomics studies provide relative quantification, they express the change in the amount of one Protein in the context of a second cellular state (for example Dunkley, Mol Cell Proteomics (2004); Hoang, J Biomol Tech 14 (2003), 216-233, Ong, Mol Cell Proteomics (2002), 376-386).

however Ultimately, the ultimate goal must be the cellular levels of absolute proteins, whether as molarity or as number of molecules per cell. The absolute quantification, which is one of the biggest challenges in proteomics based on well-established principles in analytical chemistry, and requires either external standards or internal standards (Sechi, Curr Opin Chem Biol 7 (2003), 70-77; Julka, J Proteome Res 3 (2004), 350-363). External standardization is typified by immunodetection, whether in liquid phase or on positionally addressable antibody arrays (Walter, Trends Mol Med 8 (2002), 250-253; Lopez, J Chromatogr B Analyt Technol Biomed Life Sci 787 (2003), 19-27). The second approach, which is based on internal standardization, based on mass spectrometry (MS), whereby a highly selective detection of ions (or ion fragmentation), which are characteristic of the analyte of interest, with the use of internal standards combined.

In the most meaningful Most rigorous MS analyzes will be stable isotopic variants of the analytes used as internal standards. The key of the underlying Principle is that the determination of relative signal intensities during a Mass spectrometric analysis in absolute quantities of the analyte Reference to an authentic standard, in known quantities can be converted. The direct registration This approach to intact proteins is impractical, and usually becomes the principle of surrogate applied, that means indirectly through Reference to a proteolytic peptide derived from the subject of interest Protein is derived to quantify.

analysis, based on these principles were called "AQUA" (absolute Quantification) using internal standards that de novo synthesized by chemical processes (Gerber, PNAS 100 (2003), 6940-6945). However, leads This approach itself is not good for absolute quantification of huge Numbers of proteins because each Q-peptide is chemically synthesized and independent would have to be quantified.

International Patent Application PCT / US03 / 17686, published as WO 03/102220 provides methods to determine the absolute amount of proteins present in a biological sample. The principle of WO 03/102220 is based on the formation of an ordered array of differentially isotopically labeled pairs of peptides, each pair representing a single protein, a specific protein isoform or a specifically modified form of a protein. One element of the peptide pair is a synthetically formed, external standard, and the other element of the pair is a peptide produced by enzymatic cleavage of the proteins in a sample mixture was formed. To the procedure of WO 03/102220 The standard peptides are calibrated so that absolute amounts are known and added for comparison and quantification. A sample of interest is also labeled with the same isotope label as used for the standard peptides, except that they differ in isotopic labeling. The pairs of signals corresponding to differentially labeled probes and standard peptides are finally observed and related to a list of expected masses based on the particular included standard peptides. The disadvantage of WO 03/102220 is that the standard peptides must be individually synthesized, purified and quantified. In addition, the sample and standard peptides must be separately labeled separately, increasing the potential for variability between experiments.

The WO 2004/013636 discloses a compound having an alkyl epitope tagging site and a protease cleavage site and a method for simultaneously identifying and determining expression levels of cysteine-containing proteins in normal and abnormal cells. First and second protein samples and first and second peptide samples are prepared and reacted with the disclosed compound, a proteolytic cleavage step is performed, and an affinity purification step occurs.

The WO 03/102220 provides methods for determining the absolute amount of proteins present in a biological sample. An ordered array of differentially isotopically labeled pairs of peptides is provided. One element of the peptide pairs is a synthetically formed, external standard, and the other element of the pair is a peptide formed by enzymatic cleavage of the proteins in a sample mixture.

The US 2002/0037532 discloses a method for protein identification in complex mixtures which utilizes affinity selection of constitutive peptide proteolytic fragments unique in a protein analyte. These peptides are also referred to as "signature peptides" that act as analytical surrogates. With the help of mass spectrometric analyzes of the proteolytic mixture, a protein in a complex sample can be identified without purifying the protein.

Consequently There is still an existing need, simple and easy method to be used for to develop the absolute quantification in proteomics.

Summary of the invention

• (a) mindestens zwei aufeinander folgende Peptide, die durch eine Schnittsequenz zum Trennen der Peptide verknüpft sind;
• (b) eine einzelne Markierung auf einem oder mehreren Peptid/en zur Bestimmung der absoluten Menge des Proteins; und
• (c) N-terminale und C-terminale Verlängerungen zum Schutz, zur Reinigung und zur Quantifizierung der Peptide;

wobei jedes Peptid ein einziges Protein der Probe, der Zelle oder des Organismus darstellt, das Peptid innerhalb der Reihe von Q-Peptiden einzigartig ist und jedes Peptid in einer definierten Stöchiometrie vorliegt, und wobei das Protein 20-60 Peptide umfasst.The present invention is directed to an artificial protein produced by a gene design de novo comprising concatamers of Q-peptides which are signature peptides produced by any proteolytic or chemical fragmentation for the quantitative analysis of the proteome of a sample, a Cell or organism comprising:

• (a) at least two consecutive peptides linked by a cut sequence to separate the peptides;
• (b) a single label on one or more peptide (s) to determine the absolute amount of the protein; and
• (c) N-terminal and C-terminal extensions for protecting, purifying and quantifying the peptides;

wherein each peptide represents a single protein of the sample, cell or organism, the peptide is unique within the series of Q-peptides, and each peptide is in a defined stoichiometry, and wherein the protein comprises 20-60 peptides.

To the Purpose of the invention is the artificial Protein also called QCAT protein, and the peptides responsible for the QCAT Protein are referred to as Q-peptides.

The Cleavage sequence between two Q-peptides can be an enzymatic or a chemical cleavage sequence.

The N-terminal and C-terminal extensions protect the Quantification peptides before processing and exoproteolysis. The artificial one Protein closes Features that allow easy purification of the QCAT protein.

Each of the Q peptides represents a single protein of the sample, cell or organism, and each peptide is in a defined stoichiometry which is typically, but not exclusively, 1: 1.

The The present invention further relates to a collection of Q-peptides, the complete one Cover the proteome of an organism. This collection allows a fast Quantification of the proteome of such an organism.

The The present invention also relates to a vector containing the QCAT protein and a kit comprising the vector and / or the QCAT protein.

• (a) Quantifizieren der absoluten Menge des künstlichen Proteins nach einem der Ansprüche 1–10, das Konkatamere aus Q-Peptiden und N-terminale und C-terminale Verlängerungen zum Schutz, zur Reinigung und zur Quantifizierung oder ein Peptid umfasst, das die einzelne Markierung enthält;
• (b) Erzeugen einer Präparation des zu quantifizierenden Proteins;
• (c) Mischen der Produkte der Schritte (a) und (b);
• (d) vollständiges Schneiden des künstlichen Proteins nach einem der Ansprüche 1–10 und des in Schritt (b) zu quantifizierenden Proteins an der Schnittsequenz;
• (e) Bestimmen der Masse der Peptide und daraus;
• (f) Berechnen der absoluten Menge jedes Proteins,

wobei das künstliche Protein und/oder die Peptide isotopisch markiert sind.In addition, the invention is directed to a method for the quantitative analysis of the proteome of a sample, a cell or an organism, comprising the steps:

• (a) quantifying the absolute amount of the artificial protein of any one of claims 1-10 comprising Q-peptide concatamers and N-terminal and C-terminal extensions for protection, purification and quantification, or a peptide encoding the single label contains;
• (b) generating a preparation of the protein to be quantified;
• (c) mixing the products of steps (a) and (b);
• (d) completely cutting the artificial protein of any one of claims 1-10 and the protein to be quantified in step (b) on the cutting sequence;
• (e) determining the mass of the peptides and therefrom;
• (f) calculating the absolute amount of each protein,

wherein the artificial protein and / or the peptides are labeled isotopically.

It It is important to note that the proteins are not purified need - a partial Cleaning, followed by high-resolution Separation technologies would be just as acceptable.

Brief description of the figures

1 shows examples of Q peptides selected as signature peptides;

2 shows the DNA sequence, the translated protein sequence and features of QCAT;

3 shows the characterization of the QCAT protein and Q peptides;

4 shows the quantification using the QCAT protein, and

5 shows the use of QCAT for muscle protein quantification.

Detailed description the invention

The Inventors describe the design, expression and use herein of artificial Proteins, the concatamers of tryptic Q peptides for a series of proteins that are generated by gene shaping de novo. The artificial one Protein, a Q-peptide Conkatamer ("QCAT") is designed to be both N-terminal and C-terminal Renewals includes. The function of the extensions is to protect the true Q peptides, a purification tag (such as a His tag) and a single Cysteine residue for to introduce the quantification of the QCAT.

The new gene is transformed into an expression vector that is used to express large quantities capable of being incorporated and expressed in a heterologous expression system, expressed as E. coli. Within the QCAT protein, lies the Q peptide in a defined stoichiometry (typically, but not exclusively 1: 1) before, so the whole Set of concatemated Q peptides in molar terms by determination of the QCAT protein can be quantified. In addition, the QCAT protein simply in unmarked or marked form by growth of the expression strain in a defined medium containing the selected label contains educated.

The inventors have successfully designed and constructed an artificial gene that encodes a collection of concatamers of tryptic peptides (a QCAT protein) from over 20 proteins. The protein further includes features for quantitation and purification. The artificial protein was expressed in E. coli and the synthesis of the correct product was proved by mass spectrometry. The QCAT Protein is simply cleaved with trypsin; It is easy to quantify and it can be used for the absolute quantification of proteins. This strategy makes it possible to accurately and completely quantify large numbers of proteins in proteomic studies. In addition, QCAT was labeled by selective incorporation of an amino acid labeled with a stable isotope or incorporation of ¹⁵ N nitrogen atoms at each position of the protein.

• (a) mindestens zwei aufeinander folgende Peptide, die durch eine Schnittsequenz zum Trennen der Peptide verknüpft sind;
• (b) eine einzelne Markierung auf einem oder mehreren Peptid/en zur Bestimmung der absoluten Menge des Proteins; und
• (c) N-terminale und C-terminale Verlängerungen zum Schutz, zur Reinigung und zur Quantifizierung der Peptide;

wobei jedes Peptid ein einziges Protein der Probe, der Zelle oder des Organismus darstellt, das Peptid innerhalb der Reihe von Q-Peptiden einzigartig ist und jedes Peptid in einer definierten Stöchiometrie vorliegt, und wobei das Protein 20-60 Peptide umfasst.It is therefore an object of the present invention to provide an artificial protein for the quantitative analysis of the proteome of a sample, cell or organism comprising:

• (a) at least two consecutive peptides linked by a cut sequence to separate the peptides;
• (b) a single label on one or more peptide (s) to determine the absolute amount of the protein; and
• (c) N-terminal and C-terminal extensions for protecting, purifying and quantifying the peptides;

wherein each peptide represents a single protein of the sample, cell or organism, the peptide is unique within the series of Q-peptides, and each peptide is in a defined stoichiometry, and wherein the protein comprises 20-60 peptides.

In a preferred embodiment includes the artificial Protein 20-60 peptides, most preferably 60 peptides. It is possible, multi-layered To include instances of specific peptides to stoichiometry to modify.

In yet another embodiment is the cleavage sequence by a protease, preferably by Trypsin cleaved, and the single label is a cysteine residue.

Furthermore become one or more peptides of the artificial protein identical repeated at least once, preferably once or several times, a special stoichiometry between all peptide sequences.

It is preferred, an affinity tag (eg, a histidine tag) for purification of the protein include. It is particularly preferred to include the affinity tag in either of the To include N-terminal or C-terminal extension of the protein.

In another embodiment, the protein is labeled by an isotope selected from the group consisting of ¹³ C, ¹⁵ N, ² H and ¹⁸ O.

In yet another embodiment each peptide comprises between about 3 and 40 amino acids, preferably approximately 15 amino acids.

The Protein may have a molecular weight of about 10-300 kDa, preferably about 150-200 kDa, most preferably about 150 kDa, and it is preferably expressed in E. coli.

Of the Origin of the proteome, d. H. the sample of the cell or organism, is preferably a mouse, a rat, a monkey or a human, but can be from any source of protein.

In a preferred embodiment invention, the peptides provide various conformational, metabolic or modification states of the protein to all To quantify proteins that are posttranslational from one Derived protein.

Furthermore Show the petid of the artificial Proteins preferably a defined molecular weight distribution and quantitative ratios on. A mass spectrometer can be calibrated, preferably with molecular weights that are equidistant or equidistant mass spectrometry signals lead. Preferably For example, if one or more peptides are represented twice, then unambiguously Reference molecular weight for to mark the calibration.

The The invention is further directed to a collection of concatamers of Q-peptides directed as defined in claim 1 comprising the complete proteome of an organism. All expressed proteins of an organism are defined as the proteome of such an organism. This allows the rapid quantification of the proteome of such Organism.

Furthermore is the absolute quantification in the disclosure of the present invention including the protein amounts of a particular reference strain, the following can be used about the protein levels of that particular strain or similar strains under varied experimental conditions.

The Invention is also directed to a vector comprising a nucleic acid, the artificial protein and a kit containing the artificial protein and / or the nucleic acid that includes the artificial Protein encoded. About that In addition, the invention is directed to a method for quantitative analysis directed to the proteome of an organism, described in detail above is.

The The present invention will be better understood by the examples included and results with reference to the attached figures.

Detailed description the figures

1 , Examples of Q peptides selected as signature peptides.

For a series of proteins, peptides of proteins have been selected (under Using multiple criteria), called the multiple proteins in a soluble Fraction of chicken skeletal muscle were identified and they became an artificial protein, or QCAT, composed. Left side: Coomassie blue stained, SDS-PAGE analysis of soluble Chicken muscle proteins. Middle: MALDI-TOF Spectrum of tryptic cleavage of gel pieces, the chosen Correspond to protein bands. The peptide ion with an oval symbol is the peptide that is required for incorporation into the QCAT protein was selected. Right side: Mass and position of the displayed signature peptides within the generated QCAT protein. The details of the peptides are in Table 1.

2 , The DNA sequence, translated protein sequence and features of QCAT.

The DNA sequence of the synthetic gene, with the relevant cloning sites, is shown in the upper line, and the deduced amino acid sequence is shown below. The gray-labeled areas indicate the extent of the tryptic peptides, indicating the donor chicken proteins, the tryptic peptide assay (T1-T25), and the peptide mass (in DA). A non-cleavable Arg-tryptic site within the phosphoglycerate kinase (box) is included to confirm the non-cleavability of this site. The peptides (white boxes) encode the initial methionine, the N-terminal sacrificial sequence, and spacer sequences, and are not derived from the proteins of interest. The black boxes represent the sequences carrying the single cysteine residue for quantitation and the His ₆ label for purification. T1 and T2 are rescue sequences designed to protect the N-terminus of the first true Q-peptide (T3).

3 , Characterization of the QCAT protein and the Q peptides

The pEP21a / QCAT plasmid was transformed into E. coli DE3 cells, and after a period of exponential growth, expression became of the QCAT with IPTG. The cell lysates from cells before and after induction were compared on an SDS-PAGE (image slide). After solubilization of the pellet and affinity chromatography on a NiNTA column the purified QCAT protein was homogeneous and it was in solution with Split trypsin. The peptides were tested for MALDI-TOF mass spectrometry analyzed. The inserted map for tryptic cleavage is grayed out to the relative intensities of the signals given to each Peptide in the mass spectrum to indicate; Peptides that smaller than 900 Da, which come from the "rescue" parts of the QCAT, are less easy in this type of mass spectrometry analysis due to the detectable interfering ions.

4 , Quantification using the QCAT protein

The QCAT protein was labeled unlabeled (L: "light") and in a form uniformly labeled with ¹⁵ N (H: "heavy"), produced. The H and L QCAT proteins were separately purified, quantitated and mixed in various ratios prior to tryptic cleavage and measurement of peptide intensities by MALDI-TOF mass spectrometry. Section a) represents the mass spectrum for the Q peptides for adenylate kinase (GFLIDGYPR, 12 nitrogen atoms). In section b) the measured L: H ratios are relative to the mixing ratio, in a threefold series of experiments for which individual points are shown , applied. In the lower section are the data for seven peptides collected and expressed as an average ± SD (n = 18-21). The dotted line defines the line corresponding to 95% of the safety limits.

5 , Use of QCAT for muscle protein quantification.

A preparation of soluble proteins from chicken skeletal muscle on day 1 and day 27 was mixed with [ ¹⁵ N] QCAT, cleaved with trypsin and analyzed by MALDI-TOF MS. For a subset of proteins it was possible to determine the intensities of the endogenous and a standard peptide and to calculate therefrom the absolute levels (in nmol / g tissue) of each protein. Three animals were used at each time point, the error bars are SEM (n = 3). The proteins were AK: adenylate kinase, ApoA1: apolipoprotein A1, LDHB: lactate dehydrogenase B, beta trop: betatropomyosin, beta eno: betaenolase, GP: glycogen phosphorylase, ALDO B: aldolase B, TPI: triosephosphate isomerase, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, actin, API: actin polymerization inhibitor, PK: pyruvate kinase and CK: creatine kinase.

1. Design of the gene containing the Q protein Conkatamer encoded

One of the inventors' primary interests is proteomic dynamics (Prat, Mol Cell Proteomics 1 (2002), 579-591) and changes in protein expression during muscle development (Doherty, Proteomics 4 (2004), 2082-2093, Doherty, Proteomics in press (2005)). One system that shows dramatic changes during development from immediately after hatching to adult form is the protein expression of chick pectoralis skeletal muscle. Thus, for the demonstration of the QCAT kit, the inventors selected twenty chicken proteins that were previously determined to alter the level of expression in the developing skeletal muscle (Doherty, Proteomics 4 (2004), 2082-2093). A single tryptic fragment was chosen to represent each protein (a "Q-peptide"), although a peptide reproducibly formed by any proteolytic or chemical fragmentation could be used, and in this example, Q-peptide selection was based theoretical and experimental criteria. The first criterion was that the Q peptides should not have a cysteine residue because the cysteine residue could be used for quantification of the QCAT, and the absence of cysteines should prevent complex intra- and intermolecular disulfide bridge formation in the expressed protein. Second, the proteins chosen should be unique within the set of Q-peptides. Third, Q-peptides with masses between 1000 Da and 2000 Da were selected, which corresponds to the range in MALDI-TOF mass spectra, where the sensitivity of the detection is typically high and interfering signals are low. Finally, an implementation criterion was added in that the inventors selected peptides that have already been shown to show a strong signal in MALDI-TOF mass spectrometry; 75% (15 out of 20) were Arg-terminated tryptic peptides - the propensity of such peptides to give stronger signals in MALDI-TOF mass spectrometry is well documented (Brancia, Electrophoresis 22 (2001), 552-559). A final, less important, criterion was that the Q peptides should contain at least one example of an abundant and chemically stable amino acid, such as leucine or valine, because this would facilitate metabolic labeling with amino acids for the production of stably isotopically labeled Q peptides. The peptides are summarized in Table 1: Table 1: Peptides selected for the QCAT protein. Petidmasse (Da) sequence N atoms underlying protein T1 405.2 MAGK 5 Construct, saving T2 386.25 VIR 6 Construct, saving T3 1,035.52 GFLIDGYPR 12 adenylate T4 1,601.87 VVLAYEPVWAIGTGK 16 Triosephosphatisomerase T5 1176.57 NLAPYSDELR 14 Apolipoprotein A1 T6 1,193.56 GDQLFTATEGR 15 Myosin binding protein C T7 1,789.88 SYELPDGQVITIGNER 21 Alpha actin T8 1,291.67 QVVESAYEVIR 15 Lactate dehydrogenase B T9 1,390.74 LITGEQLGEIYR 16 Betaenolase T10 1,361.63 ATDAESEVASLNR 17 Alphatropomyosin T11 1,160.58 SLEDQLSEIK 12 Myosin heavy chain (embryonic) T12 1,441.68 VLYPNDNFFEGK 15 glycogen phosphorylase T13 1,489.71 GILAADESVGTMGNR 19 Aldolase B T14 1,345.65 ATDAEAEVASLNR 17 Betatropomyosin T15 1,687.5 LQNEVEDLMVDVER 19 Myosin heavy chain (adult) T16 1,748.77 LVSWYDNEFGYSNR 19 Glyceraldehyde 3-phosphate dehydrogenase T17 1,767.98 ALESPERPFLAILGGAK 21 phosphoglycerate T18 1,249.65 QVVDSAYEVIK 13 Lactate dehydrogenosis A T19 1,803.93 AAVPSGASTGIYEALELR 21 Alphaenolase T20 1,823.97 LLPSESALLPAPGSPYGR 21 Aktinpolymerisationsinhibitor T21 1,857.9 FGVEQNVDMVFASFIR 25 pyruvate kinase T22 1,991.95 GTGGVDTAAVGAVFDISNADR 25 creatine kinase T23 274.15 AGK 4 Construct, retent T24 892.42 VICSAEGSK 10 Construct, quantification T25 1,408.68 LAAALEHHHHHH 24 Construct, cleaning marker

After the candidate set was set, the Q peptides were assembled and a gene was designed which encoded the assembled Q peptides using codons for maximum expression in E. coli. At the C-terminus, an extension was added to provide a cysteine residue and a His tag purification motif (the latter was provided in this case by the vector pET21a). An additional series of amino acids have been added to the N-terminus to provide an initial methionine residue and rescue peptide which, when cleaved off, form an actual Q-peptide ( 1 ) would expose. This avoided complications due to N-formylation or removal of methionine from the N-terminus of QCAT. The transcript encoded by the initial QCAT gene was subsequently examined in silico for features such as hairpin loops, which could affect translation. When such a feature was noted, the order of the Q peptides was reversed until an acceptable mRNA structure was obtained - the sequence of Q peptides within a QCAT is not relevant to their use as quantitation standards, and the order is thus such a manipulation accessible. The gene was constructed from a series of overlapping oligonucleotides and confirmed by DNA sequencing.

2. Expression of the QCAT

The QCAT gene was engineered with restriction sites to transform it into a set of expression vectors ( 2 ) could be installed. In this case, the gene (confirmed by DNA sequencing) was incorporated into the NdeI and HindIII sites in pET21a and was initially expressed in E. coli (NovaBlue (DE3)) growing in enriched medium. After induction by IPTG, SDS-PAGE analysis confirmed expression of a protein of expected mass (~35 KDa) in a large amount. This protein was present in the insoluble fraction of the sonicated cells and was believed to be the QCAT protein present in inclusion bodies. From this preparation, we purified the QCAT protein by affinity chromatography using Ni-NTA resin, resulting in a homogeneous preparation ( 2 , Inset). The intact average mass of this protein, as measured by ESI-MS, was 33036 ± 2 Da (data not shown) compared to the predicted mass of 33167 Da for the QCAT protein, a difference of 131 Da, which coincided exactly with the loss the methionine residue from the N-terminus. The ca. 35 KDa gel band was cleaved in the gel with trypsin and analyzed by MALDI-TOF MS. All predicted QCAT peptides were readily observed in the MALDI-TOF mass spectrum, although the N- and C-terminal rescue peptide material, by design, resulted in fragments that were too small to be seen in the MALDI-TOF mass spectrum ( 3 ). Although the peptides were chosen to give good signals in the MALDI-TOF, some peptides were remarkably less intense than others. This included all of the lysine-terminated peptides, with the exception of T17, which included a non-cleavable Arg-Pro site that, although terminated by lysine, still gave a strong signal in MALDI-TOF mass spectrometry. This was particularly evident in the isoform-specific Q peptides T8 and T18 derived from lactate dehydrogenase A and B (respectively QVVESAYEVIR and QVVDSAYEVIK); the lysine-terminated peptide contained less than 10% of the intensity of the arginine-terminated peptide, suggesting that the Q-peptides should either be drawn primarily from arginine-terminated peptides, or alternatively that a step such as guanidination should be used To convert lysine residues into homoarginine residues, which increases the tendency to give strong signal (Brancia, Electrophoresis 22 (2001), 552-559). The QCAT protein was very efficiently cleaved by trypsin and there were no signs of partial proteolytic products of the Q-peptides, which of course would affect the quantification step. The inventors subsequently expressed the protein in minimal medium with ¹⁵ NH ₄ Cl as sole nitrogen source. When cleaved with trypsin, the resulting MALDI-TOF mass spectrum was of high quality and all Q peptides were detectable at the corresponding mass shift corresponding to the number of nitrogen atoms in the peptide (data not shown).

The unlabelled and ¹⁵ N-labeled QCAT proteins were then mixed in various ratios and cleaved with trypsin before the resulting bounded peptides were analyzed by MALDI-TOF mass spectrometry. The heavy and light variants of the peptides were easily recognized ( 3a ) and their intensities were measured for a series of peptides ( 3b ). In all cases, the data were of high quality, and the ratio between the proportion of material and the heavy: light ratios was linear, with a slope of one (average ± SD (n = 6) = 1.008 ± 0.008), and very high Correlation coefficients (r ² greater than 0.99 for all cases) were achieved. The combined data ( 3c ) show the data for seven peptides; the closed boundaries, defined by the 95% confidence limits, show the quality of the quantification.

summary of results

The inventors have identified this particular QCAT in the analysis of protein expression in chicken skeletal muscle on day 1 and 27 after hatching ( 4 ) used. Twelve proteins present in this preparation were also represented in the QCAT. MALDI-TOF data of the tryptic peptides were easily obtained and the changes in protein levels occurring over the first three or four weeks after hatching were determined.

Because the proteins were absolutely quantified, the inventors were able to express the proteins as nmol per g wet weight of tissue. The variance of the triplicate analyzes was low; the inventors attributed this variance to a biological rather than an analytical variance. The inventors had recently measured the amounts of seven of these proteins by 2D gel electrophoresis and densitometry, and the correlation between the quantification using both methods was 0.82 (r ² , p> 0.001). Taking into account that the two methods measure different representation of the proteome, such as charge-variant isoforms or total protein complement, and that the densitometric method is inevitably imprecise, the correlation is good.

The Inventors have the feasibility of the QCAT approach to production of a concatameric set of Q-peptides. The QCAT that designed under both theoretical and experimental considerations, was expressed in high amounts, even on minimal medium was tightened and the product was successfully purified. Because the QCAT a complete artificial Construct, the inventors did not expect it to be any would fold recognizable three-dimensional structure, and, as expected, aggregated the protein in inclusion bodies. This is an advantage because the subsequent cleaning is easier and they only resolubilize the pellet in a highly chaotropic manner Means before affinity purification needed. About that beyond the lack of a higher one Order structure of the QCAT ensure that the QCAT at least was cleaved as fast as the target proteins that quantified should be.

Use of the artificial Protein and the QCAT peptides

It There are two ways in which the concatamers are used. First, be in the direct Q-peptide method, the concatamers as an internal Standard used. The stable isotope-labeled concatamer can directly to a sample or cell preparation prior to proteolytic Be added step. Alternatively, the concatamer quantification, for some Cell systems are used to provide absolute quantification a reference strain growth under carefully defined conditions to reach - the indirect Q-peptide Approach. This reference strain can then, in stable isotope-labeled form, as an absolute quantification standard for all future proteome quantification studies using this organism. Once a Reference strain has been accurately quantified, any peptide used to over to provide a protein, instead of the limited sentence, the is used as Q-peptides and this is clearly a very attractive one Suggestion. This extends the generality of various proteomic strategies and cretches a new niche for Labeling method, such as ICAT (Gygi, J Proteoms Res 1 (2002), 47-54 and ITRAQ (Ross, Molecular and Cellular Proteomics in Press (2004)) in a comparative proteome analysis of an unknown versus a Completely quantified strain.

however The inventors note that there are many cases where this approach is not suitable, and where a stable isotope labeled concatamer themselves (the direct Q-peptide approach) represent the appropriate standard becomes. This is especially true in proteome studies using attached to biological material that has not been previously marked can be, for example, in animal tissues or in biomarker studies.

Special advantages

The Strategy that the inventors recommend is chemical synthesis every single Q-peptide, for usually in a stable isotope form, superior. While the chemical synthesis was used in one-time applications, the process of peptide synthesis is not sufficiently "clean" to get a thorough Clean the product. Second, in multilayered or Multiplexed assays quantified each peptide individually before use Need to become. After all For example, chemically synthesized Q-peptides are a limited source the repeated expression of the QCAT gene is easy. Absolute quantification with high quality can be an effective way to overcome the difficulties those with the present Procedure for the comparative proteomics are associated, regardless of whether they are based on gel analysis or on mass spectrometry. A Series of comparative studies of specific cellular systems, any reference quantified by comparison with a QCAT would not only be quantified individually, but should also be sufficient be dramatic that when the records grow, either pairwise Comparison robust and transferable between individual laboratories and stable over the time would be.

It should possible be to ionize the tendency of the peptides to take into account and to generate a good signal in the mass spectrometer. to Time will be the ion intensities not exhaustive used in the analysis of mass spectra, although there are some recent There was an intensity using knowledge-based approaches (Krause, Anal Chem 71 (1999), 4160-4165; Gay, Proteomics 2 (2002), 1374-1391; Baumgart, Rapid Commun Mass Spectrom 18 (2004), 863-868).

An additional factor to consider is the choice of precursor label. While uniform labeling with [ ¹³ C] or [ ¹⁵ N] ensures that each peptide is extensively labeled, it may be advantageous to select copeptides such that each contains the same amino acid that is subsequently used as the stable isotope-labeled precursor. Because most QCAT proteins are thought to be composed of tryptic peptides, one strategy would be one For example, [ ¹³ C ₆ ] lysine and [ ¹³ C ₆ ] arginine also ensure that most Q peptides are simply labeled, and that the mass difference between the heavy and light peptides is constant at 6 Da. In addition, unlabelled QCAT could be labeled in vitro using reagents that are favored for comparative proteomics, which enhances all of these technologies for absolute quantification.

Without to be bound by any particular theory, they believe Inventor that the number of kopeptides present in a single QCAT could be assembled through the ability is limited, heterologous expression of large proteins in a large amount to reach. In the example given here, the inventors chose 20 peptides with an average amount of 15 amino acids and an average Molecular weight of 1.5 kDa. If 100 proteins in a single QCAT would be the result recombinant protein 150 kDa in size that express easily should let. The whole Yeast proteome, from about 6000 proteins, could then within about 60 QCAT constructs are defined.

The possibility to quantify up to 100 proteins in a single construct, represents the challenge of optimal assembly of individual Q peptides in QCATs. Various criteria are conceivable. First, one would Group of Q peptides the absolute quantification of a given subcellular Fraction or a specific subset of proteins, for For example, transcription factors or protein kinases. Secondly, and perhaps more importantly, could the concatamerization by the abundance of the target proteins in the Be controlled by a cell. It would be difficult, two different proteins with very different expression levels to quantify in the same QCAT experiment and it may be preferable strongly abundant proteins in a construct that is different from that which encodes low abundant proteins.

Other Applications for QCATs are easy to imagine, especially in the broad areas clinical biology and toxicology and diagnostics. The absolute Quantification becomes a new dimension to the predictable analysis values in clinical and other biomarker monitoring systems, and she becomes absolutely stoichiometric conditions of individual proteins within a subcellular compartment or a multiprotein complex.

material and methods

Materials.

[ ¹⁵ N] H ₄ Cl (99% atomic percent excess) was provided by CK Gas Products Ltd., Hampshire, UK. Most of the reagents, unless listed here, have previously been described (Doherty, Proteomics 4 (2004) 2082-2093, Pratt, Proteomics 2 (2002), 157-160). Chickens (Layer, Hi-Sex Brown) Skeletal muscle proteins were prepared from 1 and 27 day old chickens as a 20,000 g supernatant of a 10% (w / v) homogenate (Doherty, Proteomics 4 (2004) 2082-2093; Doherty, Proteomics 5 (1989); 2005) 5, 522-533).

QCAT gene design and construction.

The Q peptides were selected for uniqueness of mass, tendency to ionize and detectability in mass spectrometry, the presence of specific amino acid residues (for example, leucine or valine), the absence of other amino acid residues (cysteine, histidine, methionine). The peptide sequences were then randomly catalysed in silico and used to direct the design of a gene and codon optimized for expression in E. coli. The predicted transcript was analyzed for RNA secondary structure, which could reduce expression, and if present, the order of the peptides was changed. N- and C-terminal sequences have been added as rescue structures that protect the assembly of actual Q-peptides from exoproteolytic attack during expression. Additional peptide sequences were added to provide an initiator methionine and a C-terminal cysteine residue for quantification. The artificial gene was synthesized de novo (from Entelechon GmbH, Germany) from a series of overlapping oligonucleotides, verified by DNA sequencing, and ligated into the NdeI and HindIII sites of the pET21a expression vector to obtain the QCAT peptide, pET21a QCAT. A His ₆ purification label was provided by fusion with the vector.

QCAT gene expression and labeling with ¹⁵ N.

The QCAT plasmid, pET21a QCAT, was used to NovaBlue (DE3) (K12 endA1, hsdR17 (r _K12 ^- _K12 m ⁺⁾ supE44, thi-1, recA1, gyrA9, relA1, lac, F '[proA ⁺ B ⁺ , lac / ^q ZM15 :: Tn10 (Tc ^R )] cells to transform with ampicillin resistance The cells were grown at 37 ° C in Luria broth, 100 μg / ml ampicillin to an OD ₆₀₀ of 0.4-0.6 and IPTG was added to 1 mM Incubation was continued for an additional five hours when cells were pelleted by centrifugation (5000 g, 4 min, 4 ° C) in 10 ml of 20 mM Tris / HCl buffer, pH 8. The cells were then sonicated (three sonicates of 30 s) on ice and centrifuged at 14,000 g for 10 min., The pellets and the supernatants of induced. Were resuspended and lysozyme was added for ten minutes at room temperature (100 μg / ml) and non-induced cultures were analyzed by 12.5% (w / v) SDS PAGE / Coomassie blue staining For ¹⁵ N labeling, cells were grown in M9 minimal medium which was prepared using [ ¹⁵ N] H ₄ Cl (20 mM) and were induced and processed as indicated above.

Purification and analysis of the QCAT protein.

The pellets from the sonicated cells were dissolved in 20 mM phosphate buffer (pH 7.5) containing 20 mM imidazole and 8 M ureas (buffer A) before being applied to a NiNTA column (GE Healthcare). After washing with 10 column volumes of the same buffer, the bound material was eluted with buffer A with increasing concentration of imidazole (500 mM). This material was desalted on Sephadex G25 "spin columns" and the mass of eluted protein was determined by electrospray ionization mass spectrometry using a Waters-Micromass Q-ToF micromass spectrometer. The mass spectra were processed using the MaxEnt I algorithm. The purified desalted protein was cleaved with trypsin and the resulting proteins were mass determined using a Waters-Micromass MALDI-TOF mass spectrometer (Doherty, Proteomics 4 (2004) 2082-2093). To determine the response of heavy and light variants of the QCAT, the purified "heavy" and "light" proteins were mixed in different proportions before cleavage with trypsin and MALDI-TOF mass spectrometry. The intensities of the [ ¹⁴ N] and [ ¹⁵ N] peptides were measured on centroid spectra.

Use of QCAT to muscle protein expression to quantify.

The supernatant fraction containing chicken soluble proteins derived from 100 mg of tissue was mixed with 290 μg [ ¹⁵ N] QCAT, quantified by protein assay, and cleaved overnight with trypsin - the QCAT was cleaved at a greater rate endogenous muscle proteins (results not shown). The experiment was repeated for three animals at each time point. Subsequently, the [ ¹⁴ N] (muscle) and [ ¹⁵ N] (QCAT) peptides were identified by mass, and their relative intensities were determined by MALDI-TOF mass spectrometry.

Mass spectrometric analysis.

The Analysis in this example was made using a MALDI-TOF Mass spectrometer performed, but this procedure is just as applicable to all other mass spectrometry Method applicable to the analysis of peptides are suitable.

references

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SEQUENCE LISTING

Claims (14)

An artificial protein produced by a de novo gene design comprising Q-peptides concatamers which are signature peptides produced by any proteolytic or chemical fragmentation for the quantitative analysis of the proteome of a sample, cell or organism, comprising: (a) at least two consecutive peptides linked by a cut sequence to separate the peptides; (b) a single label on one or more peptide (s) to determine the absolute amount of the protein; and (c) N-terminal and C-terminal extensions for protecting, purifying and quantifying the peptides; wherein each peptide represents a single protein of the sample, the cell or the organism, the peptide within half of the series of Q-peptides is unique and each peptide is in a defined stoichiometry, and wherein the protein comprises 20-60 peptides. artificial The protein of claim 1, wherein the protein consists of 60 peptides. artificial A protein according to claim 1 or 2, wherein the cut sequence is replaced by a Protease is cut, preferably by trypsin. artificial A protein according to any one of the preceding claims wherein the single label is a cysteine residue. artificial A protein according to any one of the preceding claims, wherein one or more Peptide (s) are repeated identically one or more times to one special stoichiometry between all types of peptides. artificial A protein according to any one of the preceding claims, wherein the protein is a affinity labeling for purifying the protein. artificial A protein according to any one of the preceding claims, wherein the protein is an isotope is marked. artificial A protein according to any one of the preceding claims, wherein each peptide is about between 3 and 40 amino acids, preferably about 15 amino acids includes. artificial A protein according to any one of the preceding claims, wherein the peptides have different conformational, represent metabolic or modifying states of the protein. artificial A protein according to any one of the preceding claims, wherein the peptides are a defined distribution of molecular weight and quantitative ratios exhibit. Collection of concatamers from Q-peptides, as in Claim 1 defines the complete proteome of an organism covering the rapid quantification of the proteome of such To enable organism. Vector comprising a nucleic acid containing the artificial Protein according to one of the claims 1-10 encoded. Kit comprising the vector according to claim 12 and / or the artificial one Protein according to one of the claims 1-10. Method for the quantitative analysis of the proteome a sample, a cell or an organism, comprising the steps: (A) Quantify the absolute amount of the artificial protein after one the claims 1-10, the concatamer of Q-peptides and N-terminal and C-terminal extensions for protection, purification and quantification or a peptide comprising the single tag; (b) generating a preparation the protein to be quantified; (c) mixing the products the steps (a) and (b); (d) complete cutting of the artificial Protein according to one of the claims 1-10 and the protein to be quantified in step (b) on the cut sequence; (E) Determining the mass of the peptides and therefrom; (f) calculating the absolute amount of each protein, wherein the artificial protein and / or the Peptides are labeled isotopically.